Radiosensitization effect by combination with paclitaxel in vivo, including the effect on intratumor quiescent cells.

PURPOSE: To evaluate the radiosensitization effect on solid tumors upon combination treatment with paclitaxel (TXL), including the effect on intratumor quiescent (Q) cells. METHODS AND MATERIALS: Mice bearing SCC VII or EL4 solid tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days to label all proliferating (P) cells. The mice then received gamma-irradiation with or without tirapazamine (TPZ) at various time points after TXL administration. Another group of mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors at various time points after TXL administration. Immediately after irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, the tumor cells were isolated from the solid tumors in another group of mice, and the apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN or apoptosis frequency of Q cells was then used to calculate the surviving fraction of Q cells from the regression line for the relationship between the MN or apoptosis frequency and the surviving fraction of total tumor cells. RESULTS: In both SCC VII and EL4 tumors, maximum values of mitotic index (MI) and apoptosis frequency were observed 9 and 24 h after TXL administration, respectively. However, on the whole, the apoptosis frequency for SCC VII was very low. gamma-Irradiation 9 h after TXL administration induced significant radiosensitization effects on the total cells of both tumors. Irradiation at 60 h had a more significant effect on total cells of EL4 tumor, but no significant effect on total cells of SCC VII tumor. Combined treatment with TXL induced no radiosensitization effect on Q cells in either tumor. The effect on Q cells was observed only after TPZ was administered. The HF of total cells in EL4 tumors decreased significantly 60 h after TXL administration. CONCLUSION: No radiosensitization effect upon combination treatment with TXL is induced in Q tumor cells. However, the effect on P cells is produced by irradiation at the time when the maximum values of MI are induced following TXL administration. In addition, for tumors that are susceptible to apoptosis after TXL administration alone, irradiation at the time of sufficient reoxygenation in tumors after TXL administration produces a greater radioenhancement effect on P cells.

Transcriptional dysregulation of the p73L / p63 / p51 / p40 / KET gene in human squamous cell carcinomas: expression of Delta Np73L, a novel dominant-negative isoform, and loss of expression of the potential tumour suppressor p51.

We have recently identified a second p53 -related p73L gene, also referred to as p63 / p51 / p40 / KET gene, which encodes the 2 major isoforms p73L and p51 resulting from different exon usage at their amino terminal regions. Although p73L and p51 are suspected to play oncogenic and tumour suppressive roles in mammalian cells, respectively, no evidence of linkage between the expression of these isoforms and human cancers has been reported so far. In this study, we first investigated the expression profile of p51 and p73L in various human tumour cell lines and found that a novel isoform, termed DeltaNp73L, was predominantly expressed in squamous cell carcinomas. The expression profile of DeltaNp73L/p73L/p51 in primary human skin cancer specimens showed that the expression of p51 was frequently lost (62%) but was detected in all normal skin samples. In p51-expressing skin cancers, DeltaNp73L expression was associated at a high frequency (75%) though it was not detected in normal skin tissues. Transient co-transfection data indicate the possibility that DeltaNp73L can inhibit p53-, and more preferentially, p51-mediated transactivation. These data suggest that the loss of expression of p51 and/or the expression of DeltaNp73L might contribute to the pathogenesis of human squamous cell carcinomas.

A gene that is related to SRY and is expressed in the testes encodes a leucine zipper-containing protein.

SRY-related cDNA encoding a protein with a high-mobility-group (HMG) box and a leucine zipper motif, which was designated SOX-LZ, was isolated from a rainbow trout testis cDNA library. Comparison of this cDNA with the mouse homologous cDNA isolated from a testis cDNA library exhibits an overall amino acid sequence identity of 77%, which is in striking contrast to the abrupt loss of amino acid sequence homology outside the HMG box found among mammalian SRY genes. In both rainbow trout and mice, Northern (RNA) blot analyses have revealed the presence of a testis-specific 3-kb-long SOX-LZ mRNA, and this transcript appeared coincidentally with the protamine mRNA, suggesting its expression in the germ line. A recombinant HMG box region protein encoded by SOX-LZ could bind strongly with an oligonucleotide containing an AACAAT sequence, which is also recognized by mouse Sry and Sox-5. Upon cotransfection into CHO cells, SOX-LZ transactivated transcription through its binding motif when the region including the leucine zipper motif was deleted [SOX-LZ (D105-356)]; however, the intact SOX-LZ failed to transactivate. The intact SOX-LZ could form homodimers through the leucine zipper, which resulted in inhibition of DNA binding by the HMG box, while SOX-LZ (D105-356), which was incapable of dimerization, showed specific binding with the AACAAT sequence. Thus, the repressed transactivation of the intact SOX-LZ in CHO cells was primarily attributable to the low level of DNA binding of SOX-LZ homodimers.

Induction of inflammatory cytokines in the pleural effusion of cancer patients after the administration of an immunomodulator, OK-432: role of IL-8 for neutrophil infiltration.

When OK-432, a well-known streptococcal preparation for an anti-tumour drug, was administered into the pleural cavity of patients with malignant pleurisy, a rapid and prominent leukocytosis, predominantly consisting of neutrophils, was observed in the cavity. Neutrophil infiltration usually peaked 6-9 h after OK-432 administration, and levelled down after 24 h. Prior to the neutrophil accumulation, transient but marked elevation of various inflammatory cytokine levels including IL-1 beta, TNF-alpha, IL-8 and G-CSF was observed. In particular, IL-8 levels increased more than 10-fold, while GM-CSF did not change significantly. A good correlation between IL-8 levels and neutrophil chemotactic response was observed particularly during 0-3 h. Specific neutralization or removal of IL-8 by antibody column abrogated half of the neutrophil chemotaxis, while neutralization of C5a removed around 40%. Sequential removal of IL-8 and C5a abrogated totally 80% of chemotaxis, confirming that these two factors are mostly responsible for the neutrophil chemotaxis in the pleural fluids. These results have suggested that rapid neutrophil infiltration induced by OK-432 in vivo is ascribable largely to IL-8 and in part to C5a.

[Myasthenia gravis associated with tonic pupil].

A 41 year-old female patient with acquired autoimmune myasthenia gravis (MG, IIa type) and tonic pupil (Adie's pupil) of the right side was reported. Adie's pupil was pointed out at the same time of the clinical diagnosis of MG in September, 1988. She had the solitary thymoma identified by pneumo-mediastinograph and mediastinal CT scan. Results of the positive anti-nicotinic AChR antibody titer, the positive edrophonium test and the electrophysiological examination gave unambiguous evidences of acquired autoimmune MG with thymoma. Instillations to the eyes of cocaine, pilocarpine and epinephrine, and the pupillary response to the intravenous injection of edrophonium revealed the postganglionic abnormality mainly in the parasympathetic system. There were no other overt symptoms due to dysautonomia. She refused thymectomy. Her myasthenic and pupillary signs became gradually improving without any immunosuppressive medications. It is concluded that two diseases of the patient may be probably caused by multiple organ- or tissue-specific autoantibodies, in addition to pathogenetic significance of Adie's pupil.

[Progressive hemifacial atrophy with sympathetic nerve dysfunction of central origin].

A 37-year-old unmarried man was admitted because of gait disturbance and right hemifacial atrophy. Family history was unremarkable. He had an unconscious attack at age 13 and had writer's cramp since age 15. He was thin and lipodystrophic. In reviewing his portraits, hemifacial atrophy was considered to develop in his early teens and to be progressive since then. Pigmented gum, high arched palate, mild mental retardation, pseudo-Argyll Robertson's pupil, sexual impotence, amyotrophy of the left thigh and the right calf, and a limp due to bony abnormalities was detected. Serological tests for syphilis were negative. Bone X-rays disclosed coxa-deformance. Cerebrospinal fluid. EMG, EEG, muscle biopsy and brain CT were normal. Hearing was decreased to 20-35 dB bilaterally. Plasma norepinephrine levels were 450 pg/ml in the supine position and 539 pg/ml in standing. Plasma renin activity was 5.1-5.4 ng/ml/hr. Microneurography revealed highly accentuated muscle and skin sympathetic nerve activities. Hypothermia on the feet, reduced CVR-R and decreased mydriatic response to 5% cocaine instillation were present. Intravenous infusion of norepinephrine and intradermal injection of either acetylcholine or histamine revealed normal results. In the case, sympathicotonia due to dysfunction in the central nervous system is considered to be related to the pathogenesis of hemifacial atrophy.

[Clinical analyses on moving toes in "painful legs and moving toes"].

Clinical analyses on moving toes were done through two cases of painful legs and moving toes, who had been ill for more than 5 years. Either cases had degenerated lower lumber to upper sacral spines. Clinico-physiological data suggested the presence of polyneuropathy of the sensory type. They showed the lowered skin temperature on the distal portions of lower extremities and acrocyanosis. They, in addition, showed an irregular respiration frequently associated with apnea episodes. Frequency and amplitude of moving toes were increased during Valsalva's maneuver or apnea. Further more, findings were obtained that both the muscle sympathetic nerve activity recorded microneurographically from the tibial nerve and the surface electromyographic activity on the extensor digitorum longs were increased well concomitantly by the occurrence of moving toes. The finding may relevant that moving toes, a type of dyskinesia, may be probably generated by a direct noxious stimulus to the peripheral sensory or autonomic fibers, being strongly influenced by the higher central autonomic (at brain stem?), the extrapyramidal (at corpus striatum?) or some other structures. Moving toes might be a compensatory mechanism to lessen the paucity of blood flow in the lower extremities.

[Two cases of myasthenia gravis associated with transient amenorrhea].

Two cases of acquired autoimmune myasthenia gravis (MG) presenting transient amenorrhea were reported. Case 1, 28 years old, developed blepharoptosis and generalized fatigability at the age of 20 years. She had been treated only by anti-cholinesterase. Amenorrhea appeared at the age of 26 years. Then, physical examinations showed normal secondary sexual development and moderate myasthenic features. On laboratory examinations, SLE findings such as leucopenia (1,600/mm3), biologically false positivity in the serological tests for syphilis, negative Mantoux reaction, positive anti-nuclear and -DNA antibodies, were noted. Anti-AChR antibody was highly positive (max.: 353 nmol/l). Decreased E2 (13-15 pg/ml) and progesterone (0.21-0.29 ng/ml) values in serum, elevated LH (110-160 mIU/ml) and FSH (78-90 mIU/ml) and highly reactive LH-RH loading test were consistent with the hypergonadotropic hypogonadism. Thymus pathology of thymectomy which was done during amenorrhea, showed hyperplasia. Bilateral ovarian biopsy revealed a number of arrested primordial follicles, but neither inflammatory changes nor fibrosis. Immune complexes were not localized in the ovarian biopsy. The Kaufmann's therapy aggravated myasthenic symptoms. Menstruation recurred after 13 months of thymectomy. Amenorrhea continued for 18 months. Case 2, 37 years old, has had anti-epileptic regimens since the age of 4 years. She has been highly myasthenic for 15 years and treated by thymectomy, steroid hormone, plasmapheresis and some other therapies for 10 years. Amenorrhea occurred at the age of 34 years. Sexual development was normal. Myasthenia was very severe. On laboratory examinations, anti-AChR antibody was positive (max.: 941 nmol/l). Transient elevation of serum LH (37 mIU/l) and FSH (14 mIU/l) values was observed.(ABSTRACT TRUNCATED AT 250 WORDS)